Abstract: | Objective Endothelin 1 (ET‐1) has been implicated in the pathogenesis of fibrotic and inflammatory diseases, including scleroderma. In addition to modulating vascular tone and extracellular matrix turnover, ET‐1 up‐regulates cell surface adhesion molecules including intercellular adhesion molecule 1 (ICAM‐1), which is key to cell–cell and cell–matrix adhesion and leukocyte infiltration. This study was undertaken to delineate the signal transduction pathways utilized by ET‐1 and compare them with those adopted by proinflammatory cytokine interleukin‐1β (IL‐1β) in normal and scleroderma dermal fibroblasts. Methods Protein expression induced by ET‐1 and IL‐1β on normal dermal fibroblasts, with or without signaling inhibitors, was detected by enzyme‐linked immunosorbent assay, while messenger RNA (mRNA) levels were analyzed by LightCycler polymerase chain reaction. Expression of protein kinase Cδ (PKCδ) and PKCϵ protein in normal dermal fibroblasts and scleroderma dermal fibroblasts was determined by Western blotting, and PKCϵ involvement in ET‐1 signaling was confirmed through transfection of an ICAM‐1 promoter construct into murine PKCϵ−/− fibroblasts. NF‐κB activation was confirmed via electrophoretic mobility supershift assay, and analysis of the ICAM‐1 promoter region was achieved via transfection of deletion constructs into human dermal fibroblasts. Results In normal dermal fibroblasts, ET‐1 induced ICAM‐1 mRNA and surface protein expression in a dose‐ and time‐dependent manner via both receptor subtypes, ETA and ETB; antagonism of both abolished the ET‐1 response. MEK was involved in the signaling cascade, but phosphatidylinositol 3‐kinase and p38 MAPK were not. Key to the cascade was activation of NF‐κB, achieved by ligation of either receptor subtype. PKCϵ activation led to downstream activation of MEK and, in part, NF‐κB. IL‐1β signaling required NF‐κB and MEK activation, along with activation of PKCδ. ET‐1 and IL‐1β each utilized the same ICAM‐1 promoter region and the same NF‐κB site at –157 bp. Responses to ET‐1 and IL‐1β differed in scleroderma dermal fibroblasts, with ET‐1 sensitivity decreasing and IL‐1β responses remaining intact. Expression of PKCϵ and PKCδ in scleroderma dermal fibroblasts was also altered. Conclusion The findings of this study indicate that differences in sensitivity to ET‐1 and IL‐1β in scleroderma dermal fibroblasts may be explained by altered expression of the PKC isoforms and cytokine receptors. |